Cryptobiosis a Peculiar State of Biological Organization

Home , Cryptobiosis, Desiccation tolerance

Comparative Biochemistry and Physiology Part B 128Ž. 2001 613᎐624

Review Cryptobiosis ᎏ a peculiar state of biological organization ଝ

James S. CleggU

Bodega Marine Laboratory and Molecular and Cellular Biology, Uni¨ersity of California() Da¨is , Bodega Bay, CA 94923, USA

Received 25 September 2000; received in revised form 19 December 2000; accepted 31 December 2000

Abstract

David KeilinŽ.Ž. Proc. Roy. Soc. Lond. B, 150, 1959, 149᎐191 coined the term ‘cryptobiosis’ hidden life and defined it as ‘the state of an organism when it shows no visible signs of life and when its metabolic activity becomes hardly measurable, or comes reversibly to a standstill.’ I consider selected aspects of the 300 year history of research on this unusual state of biological organization. Cryptobiosis is peculiar in the sense that organisms capable of achieving it exhibit characteristics that differ dramatically from those of living ones, yet they are not dead either, so one may propose that cryptobiosis is a unique state of biological organization. I focus chiefly on animal anhydrobiosis, achieved by the reversible loss of almost all the organism’s water. The adaptive biochemical and biophysical mechanisms allowing this to take place involve the participation of large concentrations of polyhydroxy compounds, chiefly the disaccharides trehalose or sucrose. StressŽ. heat shock proteins might also be involved, although the details are poorly understood and seem to be organism-specific. Whether the removal of molecular oxygenŽ. anoxybiosis results in the reversible cessation of metabolism in adapted organisms is considered, with the result being ‘yes and no’, depending on how one defines metabolism. Basic research on cryptobiosis has resulted in unpredicted applications that are of substantial benefit to the human condition and a few of these are described briefly. ᮊ 2001 Elsevier Science Inc. All rights reserved.

Keywords: Anhydrobiosis; Anoxybiosis; Biochemical adaptation; Trehalose; Vitriﬁcation; Water replacement hypothesis; Metabolic rate depression; p26; Molecular chaperone; Small heat shockr␣-crystallin protein

1. Introduction and brief history comes reversibly to a standstill.’ As we shall see, the difference between a ‘hardly measurable’ In 1959 KeilinŽ. 1959 , published a benchmark metabolism and one that is at a ‘reversible stand- review on ‘cryptobiosis’, a term he coined and still’ is of considerable significance. Keilin noted defined as ‘..the state of an organism when it that cryptobiosis resulted from such things as shows no visible signs of life and when its desiccationŽ. anhydrobiosis , low temperature metabolic activity becomes hardly measurable, or Ž.cryobiosis , lack of oxygen Ž anoxybiosis . or com- binations of these. I will focus chiefly on animal

ଝ anhydrobiosis. This capability has been achieved This paper was presented at the Year 2000 Great Un- by representatives of many invertebrate taxa, knowns Symposium, Cambridge, UK. U Tel.: q1-707-875-2010; fax: q1-707-875-2009. notably in embryonic cysts of primitive crus- E-mail address: [email protected]Ž. J.S. Clegg . taceans, adult rotifers, nematodes and tardigrades

1096-4959r01r$ - see front matter ᮊ 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9Ž. 0 1 00300-1 614 J.S. Clegg rComparatië Biochemistry and Physiology Part B 128() 2001 613᎐624 and the eggs or embryos of some species in the were clear ᎏ Pouchet: no organism can survive last three taxaŽ Crowe and Clegg, 1973, 1978; complete desiccation, or return to life, once all Orstan, 1998; Ricci, 1998. . Of course, many life processes have been arrested; Doyere:` such prokaryotesŽ Potts, 1994 and Potts, 1999; Seck- organisms can be revived after complete desicca- bach, 1999. , plant seeds and other propagules tion and the cessation of their life processes. The exhibit anhydrobiosisŽ Priestley, 1986; Vertucci debate attracted interest, in part because, as and Farrant, 1995; Ingram and Bartels, 1996; KeilinŽ. 1959 documented, it was related to the Chandler and Bartels, 1999; Alpert, 2000. and major discussion over spontaneous generation in even the vegetative tissues of certain higher plants the mid-1800s. In 1859 the two protagonists ap- have achieved this ability, the remarkable ‘resur- proached the Biological Society of France to eval- rection plants’Ž. Tomos, 1992; Scott, 2000 . No uate their claims. The Society agreed and ap- example of anhydrobiosis in the early develop- pointed a special seven-man commission com- mental stages of vertebrates has been published. posed of such prominent scientists as Balbiani, This is curious since so many invertebrate em- Berthelot, Broca and Brown-Sequard.´ The bryos have achieved this ability. commission submitted a 60 000 word report in Although Keilin is best known for his work on March 1860, written by Paul Broca and signed by cytochromes and cellular respiration, we are for- all seven membersŽ. Broca, 1860 . Keilin refers to tunate that he also was interested in the topic of Broca’s prose as ‘written with great eloquence this essay and described its history in such schol- and a tendency to dramatization, which is already arly fashionŽ. Keilin, 1959 . I consider some high- reflected in the motto ‘To be or not to be, that is lights of this history, which goes back approxi- the question’.’ Interestingly, the Commission did mately 300 years to the time of Antony van not take a firm position either way, although Leeuwenhoek who apparently discovered the leaning toward the views of Doyere.` Curiously, phenomenon. Using the microscope he devel- little attention was given in the report to the oped, van Leeuwenhoek noticed that dried sedi- relationship between resuscitation and the debate ment from the gutters of houses gave rise to over spontaneous generation, which was at its ‘animalcules’, as he called themŽ most likely peak in 1860. Keilin makes the remarkable point rotifers or nematodes. shortly after the addition that while these debates were at the forefront of of water. Although he did a number of simple but French science, the appearance of Charles Dar- ingenious experiments he neither claimed that win’s ‘Origin of Species’ in 1859 ‘Produced scarely the animalcules were completely desiccated nor a ripple in the Academie´ des Sciences and in did he raise the issue of their biologicalŽ meta- other learned societies of France’Ž. Keilin, 1959 . bolic. status. Keilin’s review seems to have restored interest Science moved slowly in those days and it took in the phenomenon, a major meeting was held on approximately half a century for a resurgence of cryptobiosis in 1961Ž. Grossowicz et al., 1961 , interest, this time with the involvement of such followed approximately 10 years later by a review luminaries of their day as John Turberville Need- Ž.ŽCrowe, 1971 and book Crowe and Clegg, 1973 . ham, Henry Baker and Lazzaro SpallanzaniŽ who and the published proceedings of two subsequent seems to have been interested in just about every- scientific meetingsŽ Crowe and Clegg, 1978; thing. . Much of this work focused on nematodes Leopold, 1986. . Since that time a large body of and rotifers and their ability to reversibly desic- literature has accumulated, scattered amongst a cate, often referred to as ‘resuscitation’ or ‘resur- variety of disciplines. In many cases, the authors rection’. During the last quarter of the 18th cen- do not use Keilin’s terminology and some seem tury, debates arose concerning the status of these unaware of the phenomenon, per se, despite the dried organisms. Were they dead, but resurrected relevance of their research to this topic. by restoration of their lost water? Did they never really stop living, but simply slowed, very greatly, their vital functions? Following a two decade de- 2. Terminology-more than semantics cline of interest in these issues a heated discussion developed between two French biologists, F. Keilin pointed out the ambiguities contained in Pouchet and P. Doyere,` whose results on rotifers most of the earlier terms and their definitions. He and tardigrades did not agree. Their positions also made a distinction between dormancyŽ hypo- J.S. Clegg rComparatië Biochemistry and Physiology Part B 128() 2001 613᎐624 615 metabolism.Ž. and latent life ametabolism for matters also is the definition of ‘metabolism’. It which the term cryptobiosis was proposed. At this should be appreciatedŽ. Clegg, 1986 that point I consider an issue that is central to the metabolism is not merely the presence of chemi- significance of cryptobiosis and alluded to in the cal reactions in anhydrobionts, indeed, those are Introduction, namely, whether metabolism ever inevitable at ordinary biological temperatures. It comes to a reversible standstill. I suspect Keilin seems reasonable to require that a metabolism qualified his definition by inserting ‘and when its must consist of systematically controlled pathways metabolic activity becomes hardly measurable’myŽ of enzymatic reactions, governed in rate and di- italics. in his definition. Perhaps he recognized rection, integrated and under the control of the the difficultyŽ. impossibility? of proving the cells in which they are found. An additional re- absence of metabolism by experiment, although quirement concerns the transduction of free en- he never mentioned this problem. Before con- ergy from the environment and its coupling to sidering that point further, the conditions leading endergonic processes such as biosynthesis and to cryptobiosis require attention. Keilin used the ionic homeostasis. Thus, the severely desiccated following terms and conditions: anhydrobiosis anhydrobiont is indeed reversibly ametabolic and Ž.dehydration ; cryobiosis Ž. cooling ; anoxybiosis we may conclude that there are three states of Ž.oxygen lack ; and osmobiosis Ž high salt concen- biological organization: alive; dead; and cryptobi- tration. . Not choosing to haggle over the vague- otic. ness of the latter term, I adopt Keilin’s termi- Less philosophical and more appropriate to nology here. current research, is the mechanistic basis of anhydrobiosis, how do these organisms survive the removal of virtually all cellular water, a condition 3. Anhydrobiosis that rapidly destroys non-adapted forms? We know something about the answer. 3.1. Biological significance 3.2. Mechanisms: the water replacement hypothesis Anhydrobiosis tells us something fundamental ()WRH and ïtrification about the basic nature of living systems. Consider that an organism in anhydrobiosis lacks all the dynamic features characteristic of living organ- No attempt will be made to cover adaptations isms, notably due to the lack of an ongoing underlying anhydrobiosis at all levels of organ- metabolism to transduce energy and carry out ismic organization. These are important, of biosynthesis. In that sense it is not ‘alive,’ yet course, involving such things as specialized in- neither is it ‘dead’ since suitable rehydration pro- teguments that slow water loss and provide me- duces an obviously living organism. Therefore, we chanical protection. My coverage focuses on may deduce that it is the structural organization biochemical and biophysical adaptations that of cells and organisms, rather than their dy- seem to be of general importance rather than namics, that represents the most fundamental organism-specific. In addition, I note that this will feature of living systems. However, to draw that not be a detailed review of the large body of conclusion one must prove that severe dehydra- literature on the subject, but reviews and selected tion does indeed reversibly ‘stop metabolism.’ I papers will be cited that allow ready access. have previouslyŽ see Crowe and Clegg, 1973; A generality emerging over the last three Clegg, 1986. given reasons why one is compelled decades is the central involvement of high con- to conclude that the removal of all but, say, 0.1 g centrations of various polyhydroxy compounds, r HO2 g dry weightŽ easily achieved by anhydro- sometimes referred to as ‘compatible solutes’ bionts. , will inevitably result in the cessation of Ž.Yancey et al., 1982; Somero and Yancey, 1997 . metabolism. For example, one can calculate that Disaccharides play prominent roles, trehalose in this amount of water is insufficient to hydrate microbes, animals and lower plantsŽ Yancey et al., intracellular proteins, without which a metabolism 1982; Vertucci and Farrant, 1995; Ingram and is obviously not possible. If such a desiccated Bartels, 1996; Chandler and Bartels, 1999; Alpert, organism has been adapted for this journey, it is 2000.Ž and sucrose in higher plants Crowe and anhydrobiotic, if not, it is dead. Central to these Clegg, 1973; Elbein, 1974; Majara et al., 1996; 616 J.S. Clegg rComparatië Biochemistry and Physiology Part B 128() 2001 613᎐624

Potts, 1994, 1999; Behm, 1997; Crowe et al., 1997; complete desiccationŽ Clegg and Drost-Hansen, Goddijin and van Dun, 1999; Alpert, 2000. , al- 1990.Ž , cycles of desiccation-rehydration Morris, though other compounds are involved to a lesser 1971. and when dry survive the conditions of extent. In general, these sugars protect macro- outer spaceŽ. Gaubin et al., 1983 . Trehalose is molecules and membranes against the destructive clearly involved with the extraordinary stability of effects of water removal by replacing the primary these embryos in the face of severe environmen- water of hydration and through the formation of tal stress. Later I consider their impressive abili- amorphous glassesŽ. vitrification . These mecha- ties to undergo anoxybiosis. nisms are not mutually exclusive and the evidence The ability of trehalose and sucrose to form indicates that both are of adaptive importance in amorphous glasses at low water contentsŽ vitrifi- anhydrobiosisŽ Crowe et al., 1998a; Sun and cation. is a significant characteristic of these sug- Leopold, 1997. . arsŽ Crowe et al., 1996, 1998a; Sun and Leopold, Earlier studies revealed that trehalose was pre- 1997. although there is some discussion about the sent in high concentrations in the dormant stages extent to which this occurs in vivoŽ see Reid, of various organisms, but not in their active life 1998. . Of major importance is the enormous history stagesŽ Clegg and Filosa, 1962; Birch, slowing of the diffusion coefficients of metabolites 1963. , however, the full significance of that corre- and macromolecules present in these glasses and lation was not realized at the timeŽ. Clegg, 1965 . it is possible that their translational motion could During the 1960s two seminal groups of observa- essentially ceaseŽ. Potts, 1994, 1999 . Vitrification tions were made, one by Sydney Webb who taking place in anhydrobiotic forms would be of showed that the addition of certain polyhydroxy major importance in the avoidance of deleterious compounds could protect non-adapted bacteria interactions between intracellular components against death due to desiccationŽ. Webb, 1965 that would otherwise take place as water is re- and the other by Donald Warner whose molecu- moved, something referred to previously as ‘prox- lar modeling suggested that some of these same imity effects’Ž. Clegg, 1974 . Ž.or similar molecules might ‘fit’ the hydration An important feature of the WRH that has not lattice of proteins and possibly other large been emphasized is that the same mechanisms moleculesŽ. Warner, 1962 . The modeling was that preserve cell structure, also prevent their primitive compared with current methodology, yet, functionŽ. Clegg, 1974, 1986 . In the case of en- when combined with Webb’s results and the bio- zymes, that is of critical importance since uncon- logical correlation between dormant stages and trolled catalytic activity would be disasterous over trehalose accumulation, the stage was set for the the often prolonged duration of anhydrobiosis. In next phase of study which would lead to the addition, molecules embedded in a sugarrprotein ‘water replacement hypothesis’Ž. WRH . glass should gain substantial chemical stability Research during the 1970s and 1980s added compared to their counterparts diffusing freely in substantial but indirect evidence that trehalose aqueous solution. might be serving as a water substitute in anhydrobiotic animals. Direct evidence that this can actu- 3.3. Are stress() heat shock proteins inölëd in ally take place was obtained by Crowe and col- anhydrobiosis? leagues using model systems in vitro in an extensive series of studiesŽ see Crowe et al., 1996, 1997, Extensive evidence shows that several families 1998a,b. . Evidence for its occurrence in vivo was of heat shockrstress proteinsŽ. SPs serve as also obtainedŽ. Clegg, 1986 using encysted em- molecular chaperones to assist the folding of bryosŽ. cysts of the primitive crustacean, Artemia newly synthesized proteins, protect them from franciscana, which contain approximately 15% of stress-associated denaturationraggregation, aid in their dried weight as trehaloseŽ Clegg and Conte, their renaturation and influence the final intracel- 1980. . These encysted embryos have been studied lular location of mature proteinsŽ Jakob et al., in detailŽ Persoone et al., 1980; Decleir et al., 1993; Parsell and Lindquist, 1993; Ellis and Hartl, 1987; MacRae et al., 1989; Warner et al., 1989; 1999; Feder and Hofmann, 1999; Ellis, 2000; Browne et al., 1991. and shown to exhibit pheno- Feldman and Frydman, 2000; MacRae, 2000. . In menal stress resistanceŽ Clegg and Conte, 1980; view of their critical involvement in protection of Clegg and Jackson, 1992. . They tolerate virtually cellular proteins against damage from such things J.S. Clegg rComparatië Biochemistry and Physiology Part B 128() 2001 613᎐624 617 as temperature extremes, strong oxidizing agents, life history stages correlated the accumulation of anoxia and heavy metals it would seem reason- ‘late-embryogenesis-abundant’Ž. LEA proteins able to suppose that they would also be involved with the acquisition of desiccation-tolerance dur- in the ability of organisms to reversibly desiccate. ing seed maturationŽ Vertucci and Farrant, 1995; This potential relationship is particularly difficult Ingram and Bartels, 1996; Chandler and Bartels, to resolve, so the question asked in the heading to 1999. and in the tissues of certain water-stressed this section is by no means rhetorical. What fol- angiosperms, the ‘resurrection plants’Ž Tomos, lows refers exclusively to yeast and plants where it 1992; Scott, 2000. . A substantial amount of infor- seems that virtually all of the work on this topic mation is available on these proteins and I do not has been done. To my knowledge, no study has claim to have digested more than a sampling of been published on the participation of SPs in this literature. My general impression is that LEA animal anhydrobiosis. proteins, the related dehydrinsŽ. Close, 1997 and The classic stressŽ. heat shock response is initi- othersŽ. Garay-Arroyo et al., 2000 are involved ated by abnormal proteins, resulting eventually in more with the protection of membranesŽ includ- increased SP levels andror the synthesis of new ing associated proteins. than with the classical isoforms, the usual result being the acquisition of molecular chaperoning of damaged globular pro- enhanced stress toleranceŽ Parsell and Lindquist, teins. Other roles have been proposed, including 1993. . The situation is less clear in cells adapted water binding and retention at specific intracellu- to undergo reversible desiccation, surely a major lar sites and ion-trapping to mitigate high ionic stress but complicated by several differences strength during dehydration but, in my opinion, between water loss and the other stresses listed the evidence for these possibilities is modest. above. For a start, the metabolic shut-down in The usual classes of SP families are present in dried cells, seeds for example, does not permit these organisms, of course, but they need not be them to launch a stress response. If protein de- involved in desiccation tolerance as recent studies naturation occurs during desiccation or rehydra- on a small heat shock protein in chestnut seeds tion, we can expect that a stress response will be indicateŽ. Collada et al., 1997 . Research on a launched when the hydrated cells resume LEA-like proteinŽ. hsp12 in yeast, Saccharomyces metabolism. However, it is by no means obvious cereïsiae, provides support for the view that this how one sorts out pathways leading to desiccation particular protein is involved with membrane pro- tolerance and repair from those concerned with tection during dehydrationŽ. Sales et al., 2000 in non-related aspects such as normal developmen- general agreement with results on yeast tal processes. Ž.Eleutherio et al., 1993, 1998 . Many proteins and nucleic acids are denatured What is the initial signal involved in the pro- by removal of water in vitroŽ Colaço et al., 1994; duction of these and other proteins during the Aguilera and Karel, 1997; Crowe et al., 1997; acquisition of desiccation tolerance? In plants Allison et al., 1999. , but it is essential to de- abcissic acid seems to be of major imortance in termine whether or not they are denatured during this regardŽ Vertucci and Farrant, 1995; Ingram the desiccationŽ. andror rehydration of well- and Bartels, 1996; Chandler and Bartels, 1999. . adapted anhydrobiotic organisms. That issue is of Its appearance, resulting from decreases in tissue central importance, but a literature search turned water levels, appears to initiate signal transduc- up no information on the subject. My bias is that, tion pathways that result in the synthesis of a in general, anhydrobiotic organisms prevent pro- wide variety of proteins, including the ones men- tein unfolding and other kinds of desiccation- tioned in the previous paragraph. In the case of damage, chiefly by participation of water substi- resurrection plants this process apparently begins tutes and vitrification, rather than relying heavily in the root systems which, when sufficient water is on repair by molecular chaperones. No doubt lost, produce increased amounts of abcissic acid there are exceptions to this sweeping generaliza- which are then transported to the rest of the tion. plantŽ. Scott, 2000 . Nevertheless, evidence continues to accumulate Mostly overlooked has been the potential in- on the potential involvement of certain proteins volvement of lipid-assisted protein folding in desiccation tolerance, but not necessarily as Ž.Bogdanov and Dowhan, 1999 , but it appears molecular chaperones. Thus, early studies on plant that ‘lipo-chaperones’ could be important in pro- 618 J.S. Clegg rComparatië Biochemistry and Physiology Part B 128() 2001 613᎐624 tecting membranes during desiccationrrehydra- in the heat shock response of yeast as summa- tion, perhaps operating in concert with the pro- rized above. The extent to which sugar-SP inter- tective effects of trehalose, sucrose or other sug- actions are involved in the adaptive repertoire of ars. Much effort has been devoted to understand- organisms capable of anhydrobiosis remains an ing how the structure of membrane lipids is pro- important topic for future study. tected during water removal and addition, but less attention has been given to the participation of membrane lipids themselves, including the 4. Anoxybiosis ‘chaperoning’ of membrane-associated proteins which has been demonstrated in hydrated systems It is easy to accept that anhydrobiosis and Ž.Bogdanov and Dowhan, 1999 . It seems likely cryobiosis lead to a reversible metabolic standstill that these and other membrane properties are since the amounts of liquid water required to important as anhydrobiotic organisms undergo permit metabolism are removed in both cases. desiccation and rehydration. I am not aware of But what about other forms of cryptobiosis, any studies that attempt to explore this possibility notably anoxybiosis? Is it these that prompted using, for example, membrane preparations from Keilin to include the ‘hardly measurable’ in his organisms able to undergo anhydrobiosis and definition? He apparently did not arrive at a those from non-adapted organisms. Of course, conclusion as to whether oxygen lack can bring that is easier said than done. metabolism to a reversible standstill under physi- As described in Section 3.2, trehalose or su- ological conditions of hydration and temperature. croseŽ. sometimes other sugars are critical for the Here the definition of ‘metabolism’ becomes even desiccation-tolerance of many anhydrobionts, more critical than in the other forms of cryptobio- raising the question of potential sugar-SP interac- sis since there is an abundance of water and tions in these organisms. The importance of inter- thermal energy that will favor chemistry, if not actions between trehalose and hsp104 in the heat biochemistry. shock response of yeast have been well-docu- A vast literature documents the ability of many mentedŽ Elliott et al., 1996; Iwahashi et al., 1998; multicellular organisms to undergo periods of Singer and Lindquist, 1998a,b. . Trehalose plays oxygen lack of variable duration, however, to my the dominant protective role during exposure to knowledge no animal or higher plant can com- high temperature, whereas, trehalose is rapidly plete its life cycle under strictly anoxic conditions. degraded after heat shock because it interferes It is indeed an aerobic world for all but certain with chaperoning by hsp104Ž Singer and Lindquist, microorganisms. Nevertheless, the ability of some 1998b; also Elliott et al. 1996. . Whether this animals to survive long bouts of continuous anoxia relationship holds for reversible desiccation of is impressive. That is particularly the case for a yeast is apparently not known, but seems worth limited number of ectothermal vertebrates and a studying. Relationships between water content, substantial number of invertebratesŽ Hochahchka induction of desiccation tolerance, dehydrins and and Guppy, 1987; Bryant, 1991; Hochahchka et the oligosaccharide raffinose have been studied al., 1993; Grieshaber et al., 1994; Hand and Hard- using wheat embryosŽ. Black et al., 1999 . In that ewig, 1996; Storey, 1998; Hand and Podrabsky, system, small reductions in water content induce 2000; Jackson, 2000. . Well-adapted animals re- desiccation tolerance by starting processes in spond to anoxia by reducing their overall which dehydrins might participate, but not metabolic rates, commonly to between 1᎐10% of through interactions with raffinose. In resurrec- aerobic levelsŽ Storey, 1998; Guppy and Withers, tion plants initial dehydration leads to the pro- 1999; Hand and Podrabsky, 2000. . This response, duction of large amounts of sucrose, arising from called metabolic rate depressionŽ. MRD , has been starch, photosynthesis or the 8-carbon sugar octu- well-studied in sessile intertidal invertebrates and lose, depending on the speciesŽ. Scott, 2000 . In- involves some interesting modifications in inter- terestingly, rehydration leads to the reversal of mediary metabolism that are of obvious adaptive these pathways. One wonders whether there are significance. antagonisms between high concentrations of su- We may ask whether any of these organisms crose and the stress proteins present in cells when actually bring their metabolism to a reversible they are rehydrated, comparable to the situation standstill under anoxia, while fully hydrated and J.S. Clegg rComparatië Biochemistry and Physiology Part B 128() 2001 613᎐624 619 at physiological temperatures. The embryos of named p26, restricted to the encysted embryo certain crustaceans are good candidates. Exten- stage of the life historyŽ Jackson and Clegg, 1996; sive studies on the encysted gastrula embryos of Liang and MacRae, 1999. . We also showed that the branchiopod crustacean, Artemia franciscana, p26 underwent extensive stress-induced transloca- revealed no evidence of an ongoing metabolism tion to nuclei and other sitesŽ. Clegg et al., 1994 during bouts of anoxia that lasted for periods of and that these exhibited strong pH dependance yearsŽ Clegg, 1997; Warner et al., 1997; Clegg et Ž.Clegg et al., 1995 in a fashion consistent with al., 1999. . Recognizing that one cannot prove the intracellular pH changes in vivoŽ see Hand and absence of a rate by experiment, the case was Hardewig, 1996; Hand, 1998; van Breukelen and made that, if metabolism was occurring at all, it Hand, 2000; van Breukelen et al., 2000. . Subse- would have to be at least 50 000 times slower than quent studyŽ. Liang et al., 1997a,b showed that the aerobic rateŽ. Clegg, 1997 . Are encysted p26 belonged to the small heat shockr␣-crystallin Artemia embryos unique in this regard? Other family of proteinsŽ. de Jong et al., 1998 and candidates include copepod embryos whose demonstrated that this protein exhibited molecu- longevities in the anoxic benthos of marine lar chaperone activity in vitroŽ. Liang et al., 1997a Ž.Ž.Marcus et al., 1994 , brackish Katajisto, 1996 and probably in vivoŽ. Liang and MacRae, 1999 . and fresh waterŽ. Hairston et al., 1995 habitats, Importantly, chaperone activity in vitro did not exceeded 10 years in all three studies and, in require ATP or GTP, allowing us to suggest that certain cases, possibly centuriesŽ Hairston et al., p26 maintained protein stability during anoxia, 1995. . Long-term anoxybiosis of a nematode which provided at least some explanation for the species has been reported in which metabolic rate stability of proteins and survival of anoxic em- was reduced to undetectable levels for over 3 bryos in the ‘absence’ of metabolism. months of continuous anoxiaŽ Crowe and Cooper, Although the participation of p26 as a major 1971. . Fresh-water sponge gemmules subjected to biochemical adaptation is now established, we anoxia at room temperatures in water survived were troubled by the exception noted above and almost 4 months of continuous anoxia and most continued to seek the existence of some sort of of these were still alive when the study was termi- ongoing metabolic activity in anoxic embryos. A natedŽ. Reiswig and Miller, 1998 . It is difficult to hint came from earlier work on their guanine explain these longevities without invoking com- nucleotide poolŽ. Stocco et al., 1972 , which is plete anoxybiosis and I suspect that this pheno- unusually large and diverseŽ. Warner, 1992 , sug- 14 menon may be more common than currently gesting that one of these, Gp4 GŽ P ,P -diguano- believed. sine 5Ј-teraphosphate. , might be utilized during But there is a troublesome aspect about a anoxiaŽ. Stocco et al., 1972 . We recently re-ex- metabolic standstill during anoxybiosis. If that is amined this possibility and now have good evi- true, this would represent an exception to a major dence that this nucleotide is indeed metabolized generality of biology, namely, that a constant flow during prolonged anoxia, albeit extremely slowly of free-energy is required to maintain cellular Ž.Warner and Clegg, unpublished results . A integrity, involving biosynthesis and homeostasis. metabolic pathway leading to production of GTP

Are these organisms actually exceptions and, if Ž.and ATP from Gp4 G has been proposed, based so, then how do they accomplish that extraordi- on these results and substantial evidence from nary feat? In attempting to answer this question previous workŽ. Warner, 1992 . Thus, it appears in Artemia embryos, we examined the stability of that the great majority of intermediary proteins during long term anoxia and found no metabolism is indeed brought to a reversible evidence for protein unfolding and aggregation standstill by anoxia, but that a specialized and over years of continuous oxygen lack, in fully limited guanine polynucleotide pathway continues hydrated embryos at room temperatureŽ Clegg, to provide free energy for these embryos during 1997; Clegg et al., 1999. . That was particularly anoxia. At this point it appears that these em- surprising in view of the absence of detectable bryos might not violate the ‘free energy rule’ after protein synthesis in anoxic embryos and the labil- all, although they seem to come very close. ity of hydrated globular proteins, in generalŽ Som- Now another problem arises, what are the ero, 1995. . During the early phases of this work processes in anoxic embryos that require andror we found massive concentrations of a protein, use free energy? Curiously, the answer is not 620 J.S. Clegg rComparatië Biochemistry and Physiology Part B 128() 2001 613᎐624 obvious. Protein biosynthesis demands large per- and Higgins, 1967.Ž. . As mentioned Section 3 centages of the free energy budget of cells in trehalose became an important focus in the early general but, as described above, we are confident 1970s and the Crowe laboratory turned its atten- that this is not taking place during anoxia. An- tion to the involvement of that sugar in nematode other major free energy requirement of cells in- anhydrobiosisŽ. Crowe and Clegg, 1978 . The next volves ionic and cell volume homeostasis, but phase extended these findings by showing that encysted embryos have avoided this by becoming this disaccharide stabilized liposomes and other impermeable to non-volatile moleculesŽ see Clegg membrane preparations, as well as proteins and Conte, 1980. and, of course, by acquiring the against dehydration damage in vitroŽ see Crowe ability to undergo anhydrobiosis. et al., 1997, 1998b; Allison et al., 1999. . Many We recently uncovered a potential free energy laboratories have been actively studying these and requiring candidate, namely, p26 and its nuclear other aspects of ‘trehalose protection’ and I do translocation. Although not requiring ATP or not wish to minimize these important contribu- GTP for molecular chaperone activity in vitro, tionsŽ examples are Aguilera and Karel, 1997; that might not be the case in vivo, particularly for Collada et al., 1997; Sun and Leopold, 1997; the extensive nuclear translocation that this pro- Eleutherio et al., 1993, 1998; Reid, 1998; Koster tein undergoes during anoxia and other imposed et al., 2000. . But I think it is fair to say that the stressesŽ. Clegg et al., 1994, 1999 . Pursuing that Crowe laboratory has led the way on a relentless possibility, we recently found that p26 is a GTP- quest to answer the question ‘how does trehalose binding protein based on intrinsic fluorescence work?’ More recently, their research resulted in spectroscopyŽ Viner and Clegg, unpublished re- the use of trehalose to reduce chilling injury in sults. and have demonstrated that this protein is bovine oocytesŽ. Arav et al., 1996 , enhance recov- also a GTPaseŽ Warner and Clegg, unpublished ery and preserve function in human pancreatic results. . Our working hypothesis is that anoxic isletsŽ. Beattie et al., 1997 and to greatly increase embryos undergo a massive and almost complete the shelf-life of human blood plateletsŽ Tablin et metabolic shutdown during anoxia, but have to al., 2000.Ž including their lyophilization personal support the energetic requirements of their communication from John Crowe. . These con- molecular chaperone, p26, which they achieve by tributions are obviously of enormous economical mobilizing Gp4 G and eventually producing GTP and clinical importance. This brief account repre- andror ATP. Of course, there may be other sents a good example of the value of basic sci- endergonic processes in anoxic embryos of which ence, indeed this section might be subtitled ‘from we are unaware. tardigrades to platelets and beyond’. Others have used trehalose to improve substan- tially the survival of cryopreserved mammalian 5. Some surprising applications cellsŽ. Eroglu et al., 2000 and even to enable their reversible desiccationŽ. Guo et al., 2000 . The lat- The history of science reveals that well-ex- ter result is astonishing and supports the idea ecuted basic research sometimes results in com- expressed here and elsewhereŽ Crowe et al., pletely unpredictable outcomes that prove to be 1998b. , that the achievement of anhydrobiosis of substantial importance to the human condi- during evolution seems to have been a remark- tion. The study of cryptobiosis provides such ex- ably simple process, simply select the best ‘water amples. substitute’ and synthesize it in concentrations Nature has exploited the properties of tre- sufficient for the purpose. halose to achieve anhydrobiosis by what appears to be a remarkably simple processŽ. Section 3 , select a good water substitute and produce it in 6. Future prospects amounts sufficent to protect structure but prevent function. Some of the early work leading to that The statement just made needs further critical realization was done in the mid-1960s by John study, is it really that simple? Although possible, Crowe and associates who studied the ability of particularly in view of the results on mammalian an interesting but little-studied group of animals, cell desiccation, one would like to know more the tardigrades, to achieve anhydrobiosisŽ Crowe about the details. Anoxybiosis presents the chal- J.S. Clegg rComparatië Biochemistry and Physiology Part B 128() 2001 613᎐624 621 lenge of evaluating the significance of extremely Alpert, P., 2000. The discovery, scope and puzzle of low metabolic rates and the long-term survival of desiccation tolerance in plants. Plant Ecol. 151, 5᎐17. eukaryotic cells and organisms with such minimal Arav, A., Zeron, Y., Leslie, S.B., Behboodi, E., Ander- metabolic support. I think that increased atten- son, G.B., Crowe, J.H., 1996. Phase transition tem- tion should be given to organisms living part of perature and chilling sensitivity of bovine oocytes. ᎐ their life history in anoxic environments since Cryobiology 33, 589 599. these are good candidates to achieve complete Behm, C.A., 1997. The role of trehalose in the physi- ᎐ anoxybiosis. The utility of trehalose, sucrose and ology of nematodes. Intern. J. Parasitol. 27, 215 229. other sugars in the preservation of macro- Beattie, G.M., Crowe, J.H., Lopez, A.D., Cirulli, V., molecules, cells and tissues from non-adapted Ricordi, C., Hayek, A., 1997. Trehalose: a cryopro- organisms deserves and is receiving intense ex- techtant that enhances recovery and preserves function of human pancreatic islets after long-term amination. Largely overlooked thus far, is the storage. Diabetes 46, 519᎐523. value of some cryptobiotic organisms as useful Birch, G.G., 1963. Trehaloses. Adv. Carbo. Chem. 18, model systems for the investigation of research 201᎐226. areas of a more general nature, such as the basic Black, M., Corbineau, F., Gee, H., Come,ˆ D., 1999. mechanisms involved in the regulation of Water content, raffinose and dehydrins in the induc- metabolism, study of the physical properties of tion of desiccation tolerance in immature wheat intracellular water and the stability of biological embryos. Plant Physiol. 120, 463᎐471. materials in situ. Finally, further thought about Bogdanov, M., Dowhan, W., 1999. Lipid-assisted pro- what cryptobiosis can tell us about the basic na- tein folding. J. Biol. Chem. 274, 36827᎐36830. ture of cellular life seems worthwhile. One won- Broca, P., 1860. Rapport sur la question soumise` a la ders what Antony van Leeuwenhoek would have Societe´´ de Biologie au sujet de la reviviscence des to say about the progress achieved in understand- animaux desseches.´´ Mem. ´ Soc. Biol. 2, 1᎐140. ing the peculiar phenomenon he discovered some Browne, R.A., Sorgeloos, P., Trotman, C.N.A., 1991. 300 years ago. Artemia Biology. CRC Press, Boca Raton. Bryant, C., 1991. Metazoan Life Without Oxygen. Chapman and Hall, New York. Acknowledgements Chandler, J., Bartels, D., 1999. Plant desiccation. In: Lerner, H.R.Ž. Ed. , Plant Responses to Environmen- tal Stresses: From Phytohormones to Genome Reor- Research support from NSF grant MCB- ganization. Marcel Dekker, New York, pp. 575᎐590. 9807762 is greatly appreciated, as is manuscript Clegg, J.S., 1965. The origin of trehalose and its sig- preparation by Diane Cosgrove. John Crowe is nificance during the formation of encysted dormant thanked for describing as yet unpublished work embryos of Artemia salina. Comp. Biochem. Physiol. on protection of human blood platelets by tre- 14, 135᎐143. halose. I thank William Dowhan for a valuable Clegg, J.S., 1974. 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